Structure and antitumor activities of the water-soluble polysaccharides from Ganoderma tsugae mycelium

Abstract

Six water-soluble polysaccharides coded as GTM1 to GTM6 were extracted sequentially from the mycelium of Ganoderma tsugae with 0.2 M sodium phosphate buffer solution at 25, 40, and 80 °C, water at 120 °C, 0.5 M sodium hydroxide at 25 and 65 °C. The chemical structures were determined by using IR, EA, GC and ¹³C NMR. The weight-average molecular mass (M_w) was characterized by size exclusion chromatography combined with laser light scattering (SEC-LLS). The results indicated that the samples GTM1 and GTM2 were heteropolysaccharide–protein complexes with the protein content of 13.5 and 20.1%, and apparent-mean M_w of 62.8×10⁴, 81.8×10⁴, respectively. GTM3 and GTM4 contained (1→3)-β-d-glucans and (1→4)-α-d-glucans, while GTM5 and GTM6 were mainly a (1→6)-branched (1→3)-β-d-glucan. Two peaks were found in the SEC patterns of the four samples GTM1 to GTM4, representing fractions 1 composed of branched (1→4)-α-d-glucan with high M_w and fraction 2 composed of galactose, glucose and mannose with relatively low M_w. With the progress of isolation, the content of fraction 2 decreased from 90.2 to 57.1%, accompanying with enhancing antitumor activity. The polysaccharides GTM1, GTM2 and GTM3 had significantly higher antitumor activity against solid tumor Sarcoma 180 with the inhibition ratio beyond 50%. The results suggested that the effects of the moderate content of galactose and bound protein, as well as relatively lower M_w, on the improvement of antitumor activities of polysaccharides could not be negligible.

Introduction

Ganoderma tsugae, one of the famous traditional Chinese medicines, has long been used to promote health and longevity in China and other Asian countries. Recently, this fungus has attracted much attention because its polysaccharides have been demonstrated to have remarkable antitumor activities, which were manifested by enhancing the host mediated mechanisms including increasing IL-2 production and stimulation of cytotoxic T lymphocytes, NK activity and antibody production (Shiao et al., 1994, Su et al., 1999, Wu et al., 2001). However, the wild fruiting body of G. tsugae is scarce, and it usually takes several months to cultivate the fruiting body. Therefore, many attempts have been made to obtain bioactive substances, especially polysaccharides, from the mycelium cultured by submerged fermentation, which takes only 2 weeks to complete a cultivation and is easier to control the product quality (Yang et al., 2000, Yang and Liau, 1998). It has been reported that three antitumor heteropolysaccharide–protein complexes with the molecular mass between 1.0–1.6×10⁴ have been isolated from the mycelium of G. tsugae (Zhang et al., 1994). Sone, Okuda, Wada, Kishida, and Misaki (1985) have obtained a branched (1→3)-β-d-glucan with high antitumor activity from the culture filtrate of G. lucidum mycelium. In our previous work, a β-d-glucan-protein complex with molecular mass of 64.6×10⁴ was extracted from the G. lucidum mycelium (Chen, Zhang, Yu, & Zhu, 2000) Interestingly, a water-insoluble (1→3)-α-d-glucan isolated from the fruiting body of G. lucidum with alkali solution did not have antitumor activity, but its sulfated derivative showed antitumor activity (Zhang, Zhang, Zhou, Chen, & Zeng, 2000) It is noted that the correlation of structure of such fungal polysaccharides to antitumor activity is not fully understood. In particular, few data have been published on the secondary structure such as molecular mass, conformation and solution properties, which have been proved to have a significant effect on the anti-tumor activities (Calazans et al., 2000, Yoshioka et al., 1992) In this work, we attempted to investigate systematically the chemical structure, solution properties and antitumor activities of six water-soluble polysaccharides isolated from the G. tsugae mycelium.